Schizophrenia is a devastating neuropsychiatric disorder which affects approximately 1% of the population and results in serious disruption in the lives of afflicted individuals and their families. Common symptoms include delusions, conceptual disorganizations and visual or auditory hallucinations, as well as changes in affective behavior. A number of scales for the rating of symptoms and methods for ascertaining the diagnosis have been developed, including the DSM classification by the American Psychiatric Association (Diagnostic and Statistical Manual of Mental Disorders (4th edition), pp. 273-316, 1994), which have attempted to refine the accuracy of clinical diagnosis. However, it is likely that similar symptoms can result from several underlying abnormalities, and diagnosis relying solely on clinical symptoms is difficult and controversial, as well as subjective, time-consuming and costly.
The cause or causes of schizophrenia remain obscure. A defect in dopamine pathways of synaptic neuronal function is a central feature of the most widely held etiopathogenic theory (known as the Dopamine Hypothesis), with recent emphasis on the role of D4-type dopamine receptors (Taubes, Science 265:1034-1035, 1994). However, studies to date have failed to identify abnormalities in the basic receptor structure, suggesting that dysfunction may result from an alteration in the dynamic regulation of receptor activity.
Dopamine receptors are members of a large superfamily of G protein-coupled receptors which share a high degree of structural similarity while recognizing a widely divergent array of substances which affect cellular function. Recent advances in the study of these receptors, including the development of 3-dimensional structural models (Teeter et al., J. Med. Chem. 37:2874-2888, 1994), have led to the identification of key locations on the receptors which can modulate their function and which therefore may be sites of malfunction in schizophrenia (Samama et al., J. Biol. Chem. 268:4625-4636, 1993). One such critical location or xe2x80x9chot spotxe2x80x9d in the dopamine D4 receptor is a methionine amino acid residue (Van Tol et al., Nature 350:610-614, 1991).
A number of clinical and metabolic studies have documented altered methionine metabolism in individuals with schizophrenia (Kelsoe et al., J. Neurosci. Res. 8:99-103, 1982; Ismail et al., Biol. Psych. 13:649-660, 1978; Sargent et al., Biol. Psych. 32:1078-1090, 1992). For example, the administration of methionine has been shown to elicit an acute psychotic reaction in persons with schizophrenia but lacks such an effect in normal individuals (Cohen et al., Biol. Psych. 8:209-225, 1974). Conversely, administration of S-adenosylmethionine has been shown to have antidepressant benefits (Kemali et al., Biochemical and Pharmacological Roles of Adenosylmethionine and the Central Nervous System, Pergamon Press, pp. 141-147, 1979).
Recently, these findings have been integrated upon the discovery that methionine residue #313 (human D4 receptor numbering) of the dopamine D4 receptor appears to be abnormally modified in schizophrenic individuals (Deth, xe2x80x9cCompositions and Methods for Detection of Schizophrenia,xe2x80x9d WO 96/37780, the whole of which is hereby incorporated by reference herein). Methionine residue #313 is normally modified by the addition of an adenosyl group to its sulfur atom via the action of a methionine adenosyltransferase (MAT) enzyme; however, individuals with schizophrenia are known to be deficient in MAT activity (Carl et al., Biol. Psych. 13:773-776, 1978) and, thus, are expected to possess a lesser amount of the modified form of the dopamine D4 receptor. This deficiency is evident in a variety of tissues, including brain tissue and blood cells, particularly white blood cells, and is central to the biochemical diagnosis of schizophrenia.
This invention is based upon the discovery that a biochemical abnormality in phospholipid methylation associated with schizophrenia is linked to the abnormal modification of the dopamine D4 receptor and the clinical manifestations of altered dopamine neurotransmission. This discovery provides novel and empirical approaches for the diagnosis and treatment of schizophrenia and other related disorders.
This invention pertains to novel biochemical methods for assisting in the diagnosis of schizophrenia and other neuropsychiatric disorders, including, but not limited to, schizo-affective disorders, depression and dementias. The method of the present invention is based on the fact that dopamine receptor function is abnormal in individuals with schizophrenia, and upon the discoveries, described herein, that the dopamine D4 receptor is a direct participant in the methylation of membrane phospholipids and that phospholipid methylation is abnormally altered in schizophrenic individuals. Consequently, an altered membrane fluidity may play a significant role in producing the symptoms of schizophrenia.
In one embodiment of the present invention, the level of phospholipid methylation in a tissue sample, e.g., peripheral blood cells (such as lymphocytes), from an individual to be tested is measured, preferably using [14C]formic acid labelling or any similar method of labelling the methylfolate pool. Additionally, the level of contribution of the D4 receptor to phospholipid methylation in the same sample is also determined. The measured levels are then compared with corresponding levels of the same indicators from a tissue sample of a normal individual; a lower level of phospholipid methylation activity in the tested individual compared with the normal individual is indicative of schizophrenia or a related neuropsychiatric disorder in the tested individual.
A lower level of D4 receptor contribution to phospholipid methylation activity in the tested individual compared with the normal individual is indicative of schizophrenia specifically in the tested individual. Alternatively, a [3H]-methyl-methionine based method of phospholipid methylation activity determination is used, a more indirect assay.
This invention also pertains to novel methods for identifying therapeutic processes or agents for treatment of schizophrenia or related neuropsychiatric disorders using cultured cell lines transfected with the D4 receptor gene. Processes or agents identified by the methods described herein can increase the amount of phospholipid methylation to the normal level.
Furthermore, this invention also pertains to novel methods for determining the effectiveness of therapeutic processes or agents for treatment of neuropsychiatric disorders, and in particular schizophrenia. In one embodiment of the invention, the effectiveness of a therapeutic process or agent can be assessed by making an initial determination of the level of phospholipid methylation in a lymphocyte sample from an individual to be tested, administering the process or agent to be assessed, and making a subsequent determination of the level of phospholipid methylation in the lymphocyte sample from the individual. The corresponding levels of phospholipid methylation can be compared; an increase in the level of phospholipid methylation (preferably a normalization of methylation levels) indicates that the process or agent is effective for treating schizophrenia.
In a particular embodiment, the determination of the levels of phospholipid methylation is carried out using [14C] formic acid labelling of the methylfolate pool. Thus, the present invention has utility for the identification of agents and processes for use in the treatment of schizophrenia, and such agents, processes and drugs are also the subject of this invention.
Any tissues which display the schizophrenia-associated alterations in methionine metabolism are suitable for use in the methods of the present invention. Such tissues include brain tissue and red and white blood cells. Peripheral blood cells (especially lymphocytes) are particularly useful in the present invention because of their accessibility, allowing the methods of the present invention to be carried out on a blood sample from the individual, and because lymphocyte membranes contain the D4receptor.